The reagent WST‐1 was used to determine cell viability according to the manufacturer's instructions. 4 × 104 cells/well RGM1 cells were grown to confluence on a 96 well plate and incubated for 6–9 hr with the respective inhibitors. Afterwards, 10 μl WST‐1 was added to each well and 2 hr later absorbance was measured at 450 and 630 nm using the BioTek® Epoch Reader.
Epoch reader
Manufactured by Agilent Technologies
Sourced in United States
The Epoch reader is a microplate reader designed for a variety of applications in life science research and development. It provides accurate and reliable absorbance measurements across a wide range of wavelengths to support common microplate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
96 well tissue culture plate, by BD (6 mentions)
Mts reagent, by Promega (6 mentions)
Mst14, by R&D Systems (2 mentions)
Lb liquid nutrient medium, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 non radioactive cell proliferation assay mtt kit, by Promega (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Sars cov 2 s1 protein, by Sino Biological (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Speedmill plus, by Analytik Jena (1 mentions)
Balb c mice, by Janvier Labs (1 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiazole, by Qiagen (1 mentions)
Primescript 2 first strand cdna synthesis kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
22 protocols using epoch reader
1
Cell Viability Assay with WST-1
2
Comparative Efficacy of Targeted Fusion Antibodies
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Example 12
Fusion Abs of the disclosure were tested in several cell lines to compare the effectiveness of targeted fusion Abs versus non-targeted fusion Abs. Briefly, 1×10e4 cells/well (50 uL/well) of each cell line were seeded into 96-well tissue culture plates (Becton Dickinson). The next day cells were treated with 50 uL/well of each indicated Ab at the indicated concentrations. Six (6) days later, 20 uL/well MTS reagent (Promega) was added to the plates. Absorbance changes are read at 490 nm with a Biotek EPOCH reader. The results show that the Targeted Fusion Abs are more effective than the Non-Targeted Fusion Abs. (See,
In another experiment, 1.5×10e4 U266 cells/well (50 uL/well) or 1×10e4 CAPAN-2 cells/well (50 uL/well) were seeded into 96-well tissue culture plates (Becton Dickinson). U266 cells were treated the same day, while CAPAN-2 cells were treated the next day with 50 uL/well of each indicated Ab at the indicated concentrations. Six (6) days after treatment, 20 uL/well MTS reagent (Promega) was added to the plates. Absorbance changes are read at 490 nm with a Biotek EPOCH reader. The results show that the Targeted Fusion Abs are more effective than the Non-Targeted Fusion Abs. In addition, the masked Fusion Ab is less effective than the cleaved version. (See,
3
Masking Reduces IFNα-induced Cytotoxicity
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Example 14
In this experiment, the ability of the mask was shown to reduce off-target IFNα-induced cytotoxicity. Briefly, 1×10e4 cells/well (50 uL/well) of each cell line were seeded into 96-well tissue culture plates (Becton Dickinson). 50 uL/well of each indicated Ab at the indicated concentrations were added to the plates. Three (3) days later, 20 uL/well MTS reagent (Promega) was added to the plates. Absorbance changes are read at 490 nm with a Biotek EPOCH reader. For samples treated with MST14 (R&D Systems), 50 ug of Ab was incubated with 0.5 ug MST14 for 1 hr. At 37 deg. C. The results show that the CD138 Fusion Protein and the cleaved CD138 Fusion Protein are approximately 50× less potent that free IFNα on CD138 cells and the masked CD138 Fusion Protein is approximately 1000× less potent that IFNα on CD138 cells (See,
4
Cytotoxicity Evaluation of Fusion Proteins
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Example 15
Studies comparing the cytotoxicity of various cell lines using Masked versus Unmasked & Targeted versus Untargeted Fusion Proteins of the disclosure were performed using the following protocol. Briefly, 1.5×10e4 U266, H929, or OCI-My5.5 cells/well (50 uL/well) were seeded into 96-well tissue culture plates (Becton Dickinson). Additionally, 1×10e4 OVCAR3 or BCMW1 cells/well (50 uL/well) were seeded into 96-well tissue culture plates (Becton Dickinson). The U266, H929, or OCI-My5.5 cells were treated the same day, while OVCAR3 or BCMW1 cells were treated the next day with 50 uL/well of each indicated Ab at the indicated concentrations. U266, H929, or OCI-My5.5 were assayed four (4) days after treatment by adding 20 uL/well MTS reagent (Promega) to the plates and measuring absorbance changes at 490 nm with a Biotek EPOCH reader. OVCAR3 or BCMW1 cells were assayed similarly after six (6) days of treatment. The results show antigen targeting increases the anti-proliferative effects of the masked and unmasked fusion antibodies relative to their non-targeted counterparts. In addition, masking the interferon moiety reduces the fusion antibody's anti-proliferative effect relative to their unmasked counterparts in the cell lines assayed. (See,
5
Biofilm Formation Assay in Microplates
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The biofilm forming potential of the farm 1 isolates was tested in 96-well polystyrene microplates in tryptic soy bouillon (TSB, Merck KGaA, Darmstadt, Germany) according to the method of Kolari et al. (2003) [32 ] with minor modifications. The cell density per well was adjusted to 1 x 106 cfu/ml in 200 μl TSB. After incubation (30°C, 24 h), the wells were emptied and filled with 250 μl of crystal violet solution (4 g/l in 20% methanol). After 5 min of incubation, the plates were emptied, washed three times with distilled water (Nunc ImmunoTM Washer, Thermo Scientific, Waltham, USA) and the dye was extracted by addition of 300 μl of ethanol. After 60 min, the absorbance at 550 nm was measured by a microplate reader (Epoch Reader, BioTek, USA). The mean absorption of the blank was subtracted from the absorption of the isolates. The biofilm forming ability of the isolates was propotional to their mean absorption (n = 4) and classified as strong (Abs 550 nm ≥ 2.0), moderate (2.0 > Abs 550 nm ≥ 0.5), weak (0.5 > Abs 550 nm ≥ 0.1) or no biofilm formation (Abs 550 nm < 0.1).
6
WST-1 Assay for Cell Viability
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The reagent WST-1 was used to determine cell viability according to the manufacturer's instructions. In brief, Caco-2BBE cells were seeded at a density of 1 × 104 cells in each well of a 96 well-plate and were grown and treated as stated in the section “Cell culture, seeding density, and cytokine treatment.” Cells were incubated with respective cytokines for 24 h. At the end of treatment, 10 μl WST-1 were added to each well and 1 h later absorbance was measured at 450 and 630 nm using the BioTek® Epoch Reader. No decrease in viability was detected during exposure of any of the tested cytokines or their combination (Supplementary Figure 5 ).
7
SARS-CoV-2 RBD Protein Binding Assay
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The 96-well plate was coated with 250 ng of SARS-CoV-2 RBD protein and incubated at 4 °C overnight. The coated wells were blocked with 150 μL of 5% skim milk and incubated for 2.5 h at 37 °C. A total of five washing steps with 200 μL PBS–0.5% Tween 20 were performed. A total of 50 μL of VNAR solution was added to the designated wells and incubated for 2 h at 37 °C. The solution was removed, and the PBST washings were repeated. Binding was detected using a 1:3000 dilution of anti-HA antibody coupled to peroxidase (-HRP) after incubation for 1 h at 37 °C. The five washing with PBST were repeated, and TMB substrate was added to each well. The peroxidase-substrate reaction was stopped after 15 min by adding 10% HCl, and the OD450 (nm) was recorded with an EPOCH reader (BioTek, Winooski, VT, USA).
8
Crystal Violet Biofilm Eradication Assay
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A standard crystal violet biofilm eradication assay was performed with minor modifications as described previously.24 (link) This method gives the total amount of the biofilm material binding to the dye, including extracellular and cellular components. The 24-hour biofilms, formed in a 96-well tissue culture microtiter plate, were washed three times with 200 μL of sterile PBS solution and air-dried. One hundred microliters of antimicrobial composition dissolved in LB liquid nutrient medium (Invitrogen, Carlsbad, CA, USA) was added to each corresponding well, and the plates were incubated for 24 hours at 37°C. Then, the waste media was removed, and the plates were washed three times with 200 μL PBS solution, air-dried and stained with crystal violet 0.1% (in water) (Lenreactiv, St. Petersburg, Russia) for exactly 2 minutes. The stained biofilms were washed three times with 200 μL PBS solution, air-dried and solubilized with 200 μL of 95% ethanol for 1 hour. Then, the biofilm-associated dye was measured at OD570 using the Epoch reader (BioTek Instruments). Each experiment was performed in two independent assays. The minimum biofilm eradication concentration (MBEC) was assessed with MBEC90, which is the concentration of antimicrobials decreasing crystal violet binding in preformed biofilms by 90% compared to untreated control.
9
CLL Cell Viability Assay with Flu Stimulation
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Blood was obtained from CLL patients as defined by NCI96 criteria 28 [53 (link)] following a receipt of written informed consent under an IRB protocol approved by Saint Louis University. PBMCs were isolated from whole blood immediately following donation using Ficoll density gradient centrifugation. Isolated cells were plated in 96-well assay plates at a concentration of 10–50,000 cells (depend on patient cell numbers) per well in 100 µl of RPMI 1640 media with 10% FBS with or without 10 µM flu. The cells were cultured for 72 hrs, and then cell viability was determined using Promega’s CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (MTT) according to the manufacturer’s instructions [32 (link)]. Absorbance at 570 nm was recorded using a BioTek Epoch Reader (Winooski, VT). The rest of PBMCs from CLL patients were harvested and lysed for immunoblotting.
10
Reducing IFNα-Induced Cytotoxicity Using Masked CD138 Fusion Protein
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Example 14
In this experiment, the ability of the mask was shown to reduce off-target IFNα-induced cytotoxicity. Briefly, 1×10e4 cells/well (50 uL/well) of each cell line were seeded into 96-well tissue culture plates (Becton Dickinson). 50 uL/well of each indicated Ab at the indicated concentrations were added to the plates. Three (3) days later, 20 uL/well MTS reagent (Promega) was added to the plates. Absorbance changes are read a 490 nm with a Biotek EPOCH reader. For samples treated with MST14 (R&D Systems), 50 ug of Ab was incubated with 0.5 ug MST14 for 1 hr. At 37 deg. C. The results show that the CD138 Fusion Protein and the cleaved CD138 Fusion Protein are approximately 50× less potent that free IFNα on CD138 cells and the masked CD138 Fusion Protein is approximately 1000× less potent that IFNα on CD138 cells (See,
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